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Preparation method and purification method of recombinant human-derived III type collagen

A technology of collagen and purification methods, applied in peptide preparation methods, chemical instruments and methods, animal/human proteins, etc., can solve the problems of difficult product purification, low biological activity of expressed products, and difficult application of expressed products, etc., to reduce Production of endotoxin, effect of improving purity

Active Publication Date: 2017-08-11
山东多美康生物医药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Most domestic collagen manufacturers use Escherichia coli to ferment and express collagen, but bacterial expression systems generally have the following disadvantages: the pyrogens produced (such as endotoxin) make the expression products difficult to apply clinically; the target protein is often expressed in the form of inclusion bodies, causing the product Purification is difficult; the post-translational modification system of the prokaryotic expression system is not perfect, and the biological activity of the expression product is low, etc.

Method used

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  • Preparation method and purification method of recombinant human-derived III type collagen
  • Preparation method and purification method of recombinant human-derived III type collagen

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preparation example Construction

[0052] In another aspect, the present invention provides a method for preparing recombinant human type III collagen, which comprises:

[0053] Cultivate a recombinant yeast capable of expressing recombinant human type III collagen to obtain a fermentation broth;

[0054] The above fermented liquid is purified according to the above-mentioned purification method of recombinant human type III collagen to obtain recombinant human type III collagen.

[0055] The recombinant human type III collagen produced by the above preparation method has high purity and high activity, reaching the level of high-purity collagen that meets cosmetic raw materials, medical equipment and medical grade.

Embodiment 1

[0058] The purification method of recombinant human type III collagen provided in this example includes the following steps.

[0059] 1.1 Filtration steps:

[0060] Take the fermented broth fermented by recombinant yeast (preservation number CGMCC No. 12491) capable of expressing recombinant human type III collagen, centrifuge at 5000 rpm for 10 min; use filter paper (general laboratory qualitative filter paper) for the supernatant Filter and collect the filtrate.

[0061] CGMCC No.12491 The cultivation method of the recombinant yeast refers to the application number 2016111191228, and the cultivation method in the Chinese patent application named "a fermentation method and fermentation medium of recombinant human type III collagen engineering bacteria" is cultivated, Obtain a fermented broth.

[0062] Biological deposit information of the recombinant human type III collagen engineered bacteria: classified as Pichia Pastoris (Pichia Pastoris), on May 23, 2016 at No. 3, Court...

Embodiment 2

[0091] This embodiment provides a preparation method for recombinant human type III collagen, which includes:

[0092] Cultivate a recombinant yeast (CGMCC No.12491) capable of expressing recombinant human type III collagen to obtain a fermentation broth; the reference application number for the cultivation method is 2016111191228, and the name is "a recombinant human type III collagen engineering bacterium" The cultivation method in the Chinese patent application of "fermentation method and fermentation medium" is cultivated to obtain a fermentation liquid.

[0093] According to the purification method of recombinant human type III collagen provided in Example 1, the collagen stock solution was obtained.

[0094] In summary, the purification method of recombinant human type III collagen provided by the present invention adopts a heat denaturation method to prevent collagen from degrading into small molecular peptides, and uses ammonium sulfate precipitation to remove impuriti...

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Abstract

The invention relates to the technical field of collagen extraction and discloses a preparation method and a purification method of recombinant human-derived III type collagen. According to the purification method of the recombinant human-derived III type collagen, by thermal treatment of fermentation liquid, obtained by fermentation of recombinant saccharomycetes capable of giving expression to the recombinant human-derived III type collagen, at a temperature of 65-75 DEG C, degradation of the recombinant human-derived III type collagen in the fermentation liquid into small-molecule peptides is effectively prevented, endotoxins in the fermentation liquid can be reduced, and accordingly purity of the recombinant human-derived III type collagen is greatly improved to meet requirements of cosmetic raw materials, medical instruments and medical high-purity collagen. The recombinant human-derived III type collagen prepared according to the method is high in purity and low in endotoxin content and meets requirements of cosmetic raw materials, medical instruments and medical high-purity collagen.

Description

technical field [0001] The invention relates to the technical field of protein extraction, in particular to a purification method and preparation method of recombinant human type III collagen. Background technique [0002] At present, the sources of collagen are mainly divided into: [0003] The traditional extraction method of animal-derived collagen: it is extracted from animal connective tissue (pig skin, cowhide, donkey, skin, fish, etc.) by acid and alkali hydrolysis, but the product components obtained are complex. [0004] Collagen-like chemical synthesis method: chemical synthesis method provides a fast and efficient protein preparation method, and it can easily introduce unnatural amino acids, change carbon chain skeleton and perform other chemical modifications to improve protein activity and construct new proteins. This technology is applied in the synthesis of collagen-like protein, and its technical characteristics are used to endow collagen-like protein with n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C07K1/113
CPCC07K14/78
Inventor 贾芳苗刘冬邓飞
Owner 山东多美康生物医药有限公司
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